Novel muscle relaxant using negative charge gold nanoparticle with choline

ABSTRACT

A drug having a muscle relaxing effect can be prepared by binding negatively charged gold nanoparticles having a size of 1.4 nm fitted to synaptic clefts to choline through ionic bonds, thereby inducing gold-sulfur bonds in the Cys loop of an acetylcholine receptor in neuromuscular junction, and thus blocking the electric current induced in signal transduction from a nerve to a muscle, thereby blocking neuromuscular transmission chemically and physically. When the reverse is administered after surgery, the gold-sulfur bonds are broken due to the re-flow of the electric current, and thus the gold nanoparticles are again released and metabolized into neuromuscular junction. Further, such muscle relaxing effect can be applied as an anticonvulsant.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Choline is a kind of vitamin B complex, and acts as a partial agonistfor acetylcholine, a neurotransmitter, in neuromuscular junction.Negatively charged gold particles are formed by adhering alkaline COO⁻particles to a thin film of gold nanoparticles. When the negativelycharged gold particles form ionic bonds with choline and then theresulting complexes are injected intravenously, the complexes migrate toan acetylcholine receptor in neuromuscular junction of a patient within1 minute, and the choline binds to an acetylcholine-binding site therebyforming ionic bonds. The migrated nanoparticles enter the extracellularvestibule of the receptor together with a high concentration of thecholine and migrate to a cysteine loop to thereby bind to the SH groupsof the cysteine due to the strong binding force of gold-sulfur ions, andsubsequently prevent from forming alpha subunit S═S bonds therebyblocking a flow of electric current through open ion gates. Accordingly,it is anticipated that the choline-gold nanoparticle complexes willperform a role of blocking the signal transmission in neuromuscularjunction more strongly.

Since the action of cholinesterase is inhibited if reverse isadministered, acetylcholine is not degraded and the acetylcholine boundto the receptor induces S═S reducing bonds in the cysteine loop. Thenegatively charged gold nanoparticles are come out of the ionic paths byrepulsion since the gold-sulfur binding force is released due to theelectric current generated by the reducing bonds.

2. Description of the Related Art

The signal transduction at neuromuscular junction is achieved bychemical means such as a neurotransmitter, or physical means such as anionic path. Prior muscle relaxant is chemical means that blocksneuromuscular transmission since it has a similar structure toacetylcholine, a neurotransmitter, and thus binds competitively toacetylcholine and its receptor. If a method of blocking the flow andmaintenance of electric current at ionic paths by using goldnanoparticles having a negative charge is used in combination with theprior means, the effect of muscle relaxing will be doubled. Further,such strong muscle relaxing effect can be applied as an anticonvulsant.

SUMMARY OF THE INVENTION

Choline

Choline is a vitamin B complex having2-hydroxy-N,N,N-trimethylethanaminium structure, and passes through thecerebrovascular barriers. It takes only 1-2 msec for acetylcholineisolated from a presynaptic terminal to work on the postsynapticmembrane. Once a postsynaptic stimulant voltage is generated,acetylcholine is hydrolyzed into choline and acetate by cholinesterase.The choline and acetate are re-synthesized into acetylcholine by cholineacetylase and are used.

Signal Transduction at Neuromuscular Junction

A neurotransmitter in a synaptic vesicle is released by a nerve impulsetransmitted from a cell body to an axon terminal. As the nerve impulsereaches a cell membrane of a terminal bouton along the axon, a calcium(Ca⁺⁺) channel present in the location is opened, and calcium ions enterthe terminal bouton, and the membrane of the synaptic vesicle is fusedwith the cell membrane.

When a presynaptic cell membrane is fused to the synaptic vesicle, theneurotransmitter in the synaptic vesicle is released into synapticclefts and binds to a receptor present in a postsynaptic cell membrane.The receptor acts as a kind of a ligand-sensitive ion channel. Thechannel is an ion channel that is opened by coupling with a ligand,i.e., the neurotransmitter. When a cation channel such as a sodiumchannel or a calcium channel is opened, a cation that was present inextracellular fluid enters the postsynaptic portion, and thus thevoltage inside the cell that has maintained a stable membrane electricpotential (−80 mV) is partially changed. As the voltage reaches athreshold (−70 mV), a voltage-sensitive sodium channel is opened and alarge quantity of the sodium ions in the extracellular fluid come insidethe cell, and accordingly depolarization occurs thereby reversing themembrane electric potential of this portion. As the membrane electricpotential is changed like this, a voltage sensitive potassium channel isnow opened. As potassium ions come outside the cell, the membraneelectric potential returns to its original status.

The sodium and potassium ions in which the outside and inside of thecell are reversed from the original status are exchanged in 1:1 ratio bya sodium/potassium (Na⁺/K⁺) exchange pump. At this time, energy isneeded.

When the ion exchange is completely finished, the membrane electricpotential returns to the original status and the membrane again becomescapable of responding to a stimulus.

The sodium ions enter a specific portion of a cell and depolarize notonly the portion but also are diffused along an axon and depolarize theadjacent portion. If the diffusion occurs toward a cell body, the sodiumchannel is inactivated and thus an impulse is not continuouslytransmitted. However, if the sodium ions are diffused toward thesynaptic terminal of the axon, the adjacent portion of the membrane isdepolarized and the active electric potential is also generated at thisportion.

Structure of an Acetylcholine Receptor

A nicotinic acetylcholine receptor is an ionic receptor controlling theionic path in a cell membrane through a ligand. The nicotinicacetylcholine receptor consists of five subunits, and each subunit isvery similar in structure. An acetylcholine receptor of a muscleconsists of two α subunits and β, γ and δ subunits. The site bindingwith the receptor in a muscle is outside α subunits near N termini. Ifacetylcholine binds to the site, the a subunits become more similar toother subunits in structure, and thus the ionic path becomes moresymmetrical and is opened to a size of about 0.65 nm.

The nicotinic acetylcholine receptor has a similar structure to a musclereceptor on signal transduction in a nerve. When acetylcholine bindscontinuously to the site, the ionic path maintains its open state. Asthe ionic path is opened, ions having a positive charge, particularlysodium and calcium ions, enter inside the cell.

Na⁺ and K⁺ are all penetrable to the nicotinic acetylcholine receptor,and in a certain site, Ca²⁺ is also penetrable. Na⁺ and K⁺ penetrate anopen site of the ionic path, and this process occurs by the change inthe structure of each subunits.

It is known that the nicotinic acetylcholine receptor consists ofextracelluar, transmembrane and intracellular domains (ICD) fromresearch through Gaussian Network Model (GNM) and Anisotropic NetworkModel (ANM). The opening and closing of the ionic path are determined bysymmetrical 3-dimensional twisting movement and asymmetrical tiltingmovement between each subunit. At this time, the pore size between pathsis determined by the opening and closing of the disulfide (S═S) bond ofcysteine (Cys) loop in and between subunits. Accordingly, if goldnanoparticles having a negative charge interrupt the disulfide bond ofCys loop between each subunit, the flow and maintenance of the electriccurrent to the neuromuscular junction site can be blocked.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically a structure in which ionic bonds areformed between choline having a positive charge and a nanoparticlehaving a negative charge;

FIG. 2 illustrates schematically a process that choline injectedintravenously after forming ionic bonds with gold nanoparticles having asimilar structure to acetylcholine binds to an amino acid of a bindingsite on an acetylcholine receptor, and the nanoparticles, in which theionic bonds are disrupted, bind to the SH groups of Cys loop byrepulsion to the binding site having a negative charge;

FIG. 3 illustrates the analysis results for choline and nanoparticles byUV spectrophotometry, showing that the light-absorbing configurations ofthe choline and the nanoparticles were similarly achieved; and

FIG. 4 illustrates the analysis results for choline-nanoparticlecomplexes by UV spectrophotometry, showing that the ionic bonds betweenthe choline and the nanoparticles were smoothly formed by confirming theintensity and the light-absorbing region of the choline-nanoparticlecomplexes were more extended than those of the respective singlemolecules;

FIG. 5 is a graph illustrating the effect of choline-nanoparticlecomplexes over time in an experiment employing Ach, and schematicallydepicting a process that immediate reversal is achieved with thereceptor inactivation effect of about 75% over time; and

FIG. 6 is a graph illustrating the effect of choline-nanoparticlecomplexes over time in an experiment employing DMPP, and schematicallydepicting a process that immediate reversal is achieved with thereceptor inactivation effect of about 75% over time.

DETAILED DESCRIPTION OF THE INVENTION

Firstly, ionic bonds are induced by vortexing and pipetting cholinehaving a positive charge and gold nanoparticles having a negative chargefor one hour. The degree of binding is determined through the wavelengthanalysis by UV VIS spectrophotometry.

FIG. 1 illustrates ionic bonds between 1 mM choline and 30 nM negativelycharged nanoparticle: the configuration that COO⁻ groups coated on athin film of the nanoparticle binds to N⁺ groups of the cholinemolecules is schematically illustrated.

FIG. 2 illustrates schematically the binding site of choline and goldnanoparticles having a negative charge on a nicotinic acetylcholinereceptor at a neuromuscular junction postsynaptic muscle cell. The redcolor depicts the extracellular domain of the receptor, the violet colordepicts the terminal of the choline molecule binding on the receptor,the blue color depicts transmembrane and intracellular domains, and thepale blue color depicts sodium and calcium ions passing through an ionicpath. When choline-nanoparticle Complexes bind strongly to the receptor,the gold nanoparticle molecules are detached from the binding body byrepulsion and block the flow of the ionic path through adsorption of theSH groups inside a glycoprotein receptor.

FIG. 3 illustrates the analysis results for choline and goldnanoparticles having a negative charge by UV VIS spectrophotometry. Theblack graph depicts the analysis results for the choline, and the redgraph depicts those for the gold nanoparticles having a negative chargeby UV VIS spectrophotometry. It was confirmed that the absorbance of thetwo molecules was achieved in a similar degree within almost the samewavelength range. The used apparatus was Shimadzu UV3101PC made inJapan. The measurement was made in a light absorbing mode in the UVwavelength range of 190 to 300 nm, and the resolution was possible to0.1 nm.

FIG. 4 illustrates the analysis results for complexes of the choline andthe gold nanoparticles having a negative charge by UV VISspectrophotometry, and shows an intensity stronger and a light absorbingrange in a wavelength region wider than the absorbance of the respectivemolecules. The used apparatus was Shimadzu UV3101PC made in Japan. Themeasurement was made in a light absorbing mode in the UV wavelengthrange of 190 to 300 nm, and the resolution was possible to 0.1 nm.

Organ Model Experiment

The left adrenal of a Sprague-Dawley white rat was removed andcannulated according to the Wakade method, and then it was extirpatedand immobilized to a leucite chamber, and an experiment was performedwhile perfusing with Krebs fluid.

A single injection of ACh (5.32×10⁻³ M) was treated in 0.05 ml of abuffer solution containing 137.5 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mMMgCl₂ and 10 mM D-glucose at room temperature for 3 minute. Then, theamperometric current generated by the secreted catecholamine wasmeasured by employing an 8-μm-diameter carbon electrode. Data wascomputerized with IGOR software and the buffer solution was perfusedevery two seconds.

The agonistic effect of the choline was confirmed with a controlexperiment employing 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP),the choline-nanoparticle complexes were treated again for 3 minutes, andthen the change in the catecholamine secretion was measured.

Finally, 10 μM DMPP stimulation was performed for 2 seconds as reverse,thereby confirming whether catecholamine secretion was restarted, andverifying whether the choline-nanoparticle complexes were released fromthe ionic path.

RESULT

FIG. 5 illustrates the effect of the choline-nanoparticle complexes overtime. When 0.05 ml of a single injection of ACh (5.32×10⁻³ M) was evokedin a rat adrenal gland, the receptor activation was confirmed after 15minutes, and then when the choline-nanoparticle complexes were treated,it was confirmed that the catecholamine secretion was decreased to about75% after 60 minutes. When Ach was again treated after about 1.5 hours,it was confirmed that immediate reversal was achieved. *: P<0.01.

FIG. 6 illustrates the results of an experiment for the effect of thecholine-nanoparticle complexes employing DMPP over time. When a singleinjection of DMPP was evoked in a rat adrenal gland, the receptoractivation was confirmed after 15 minutes, and then when thecholine-nanoparticle complexes were treated, it was confirmed that thecatecholamine secretion was decreased to about 75% after 60 minutes.When DMPP was again treated after about 3.5 hours, it was confirmed thatimmediate reversal was achieved. *: P<0.01.

By binding gold nanoparticles to choline through ionic bonds, a drughaving a stronger muscle relaxing effect obtained by blocking physicallyother ionic paths than prior muscle relaxants competitively inhibiting areceptor-ligand binding can be developed. Further, the complexes can beapplied as a therapeutic agent for epilepsy.

1. A method of developing a stronger muscle relaxant having no effect ona physiological process of the human body by a mechanism in which goldnanoparticles having an anion binds to choline through ionic bonds, sothat when the choline-gold nanoparticle complexes are injectedintravenously into a patient on surgery, the complexes migrates to anacetylcholine receptor of a muscle and then the ionic bonds of thecomplexes are broken, so that the choline binds to a receptor bindingsite as a partial agonist, and the gold nanoparticles bind to the Cysloop, thereby blocking the chemical and electrical neurotransmittance,respectively, and thus doubling the muscle relaxing effect; and when thereverse is administered post surgery, the gold-sulfur bonds are brokendue to the electric current recovered to the receptor, and thus the goldnanoparticles are released ex vivo.
 2. A gold nanoparticle as aneffective therapeutic agent for epilepsy seizure wherein the goldnanoparticle comes into an ionic path and provides a strong musclerelaxing effect with tracheal intubation for breathing on statusepilepticus seizure, and generalized and local epilepsy seizure due toits strong anti-acetylcholine action.
 3. A method of confirming whetherionic bonds are properly formed between choline and negatively chargedgold nanoparticles, wherein the method comprises: measuring theabsorbance of the choline and the negatively charged gold nanoparticlesby employing a UV spectrophotometer; and observing whether theabsorbance of the choline-nanoparticle complexes is shown as a changedconfiguration different from that of the respective single molecules. 4.Use of catecholamine secretion of large dense-core vesicles (LDCVs)secreted by acetylcholine in a bovine adrenal gland chromaffin cell inamperometric measurement for observing the anti-cholinergic action ofcholine-nanoparticle complexes.